Glioblastoma-synthesized G-CSF and GM-CSF contribute to growth and immunosuppression: Potential therapeutic benefit from dapsone,fenofibrate,and ribavirin

Abstract

Increased ratio of circulating neutrophils to lymphocytes is a common finding in glioblastoma and other cancers. Data reviewed establish that any damage to brain tissue tends to cause an increase in G-CSF and/or GM-CSF (G(M)-CSF) synthesized by the brain. Glioblastoma cells themselves also synthesize G(M)-CSF. G(M)-CSF synthesized by brain due to damage by a growing tumor and by the tumor itself stimulates bone marrow to shift hematopoiesis toward granulocytic lineages away from lymphocytic lineages. This shift is immunosuppressive and generates the relative lymphopenia characteristic of glioblastoma. Any trauma to brain—be it blunt, sharp, ischemic, infectious, cytotoxic, tumor encroachment, or radiation—increases brain synthesis of G(M)-CSF. G(M)-CSF are growth and motility enhancing factors for glioblastomas. High levels of G(M)-CSF contribute to the characteristic neutrophilia and lymphopenia of glioblastoma. Hematopoietic bone marrow becomes entrained with, directed by, and contributes to glioblastoma pathology. The antibiotic dapsone, the lipid-lowering agent fenofibrate, and the antiviral drug ribavirin are Food and Drug Administration– and European Medicines Agency–approved medicines that have potential to lower synthesis or effects of G(M)-CSF and thus deprive a glioblastoma of some of the growth promoting contributions of bone marrow and G(M)-CSF.

Keywords

Dapsone fenofibrate granulocyte-colony stimulating factor glioblastoma granulocyte–monocyte–colony stimulating factor immunosuppression lymphopenia myeloid-derived suppressor cells neutrophilia radiotherapy ribavirin temozolomide

Introduction

This article shows that normal brain tissue, damaged brain tissue, and a growing glioblastoma (GB) all communicate with bone marrow (BM). In GB, this communication enhances tumor growth. Both relative lymphopenia and elements of the immunosuppressive milieu of GB are consequences of what Stromnes et al.¹ termed “complicity of bone marrow” in nonhematopoietic cancer’s growth. This occurs in GB by both surrounding brain and the GB tissue itself synthesizing granulocyte-colony stimulating factor (G-CSF) and granulocyte–monocyte–colony stimulating factor (GM-CSF), hereafter notated as G(M)-CSF when referring to both.

This article ends by showing how three already-marketed drugs, dapsone, fenofibrate and ribavirin, can be repurposed to lower G(M)-CSF or its deleterious effects. G-CSF is a ~20-kDa glycoprotein and GM-CSF is a ~15-kDa glycoprotein, both synthesized by a variety of cells throughout the body. G(M)-CSF signals through their each specific dimeric receptors to activate intracellular Janus kinase (JAK)/signal transducer and activator of transcription (STAT)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase signaling.²

For many functions, G-CSF and GM-CSF can cross-cover. For some functions, they cannot. G(M)-CSF drives BM CD34+ hematopoietic stem cells to proliferate (symmetrically and asymmetrically), survive, and differentiate to mature monocytes, lymphocytes, and granulocytes of circulating blood.² Many cells synthesize and release G(M)-CSF, for example, glia, neurons (vide infra) macrophages, T cells, endothelial cells, fibroblasts, and importantly, some cancers including GB as discussed below.

Pharmaceutical GM-CSF is marketed as sargramostim and pharmaceutical G-CSF as filgrastim and pegfilgrastim. These are often life-saving drugs during severe neutropenia and other BM failure states.²

Normal brain tissue synthesizes G(M)-CSF

G-CSF is synthesized by normal, unstimulated brain neurons^3–5 as is GM-CSF.^3,6 G(M)-CSF plays an important role in normal brain and neuronal function, normal central nervous system (CNS) neurodevelopment, and neuronal repair after ischemia or trauma^3–9 independent of any effect on hematopoiesis or BM function. Normal resting human astrocytes also synthesize both G-CSF and GM-CSF.¹⁰ GM-CSF is neuroprotective after a wide range of neurotrauma, exerting neuroprotective effects by diminishing expression of apoptosis-related genes.^11–15

Brain tissue injury increases brain-synthesized G(M)-CSF

In 1991, it was first shown that injured mammalian brain produced factors with GM-CSF and interleukin (IL)-3 agonist activity.¹⁶ Since GM-CSF and IL-3 are known products of microglia,¹⁷ microglia were the suspected brain cell origin.

Surgical trauma promotes conditions for enhanced residual GB cell migration and growth.^18–21 Hamard et al.¹⁸ reviewed the multiple growth-enhancing mediators that are mobilized or increased in the glioma surgical field, suggesting a worthwhile target would be to “normalize and re-educate the molecular and cellular responses at the resection margin to promote anti-tumorigenic processes.”

In a murine orthotopic glioma model, post-resection margins show non-transformed but activated astrocytes that facilitate growth of the few remaining glioma cells, as evidenced by recurrent gliomas growing faster than the pre-resection gliomas.²² G(M)-CSF increase is a primary driver of this phenomenon as indicated by the following data.

Serum G-CSF is acutely elevated in experimental animal closed head trauma and in humans in the emergency room after traumatic brain injury,²³ findings confirmed in studies in humans where timing of G(M)-CSF peak in plasma parallels peak elevation at the cerebral wound site during the first days following brain injury.^24,25

Post-traumatic hypoxia in patients with closed head trauma increases GM-CSF in the cerebrospinal fluid.²⁶ Whole body radiation that included brain induced increased synthesis of G-CSF that, although it contributes to BM recovery,²⁷ contributed to systemic immunosuppression (see section “Elevated G(M)-CSF is immunosuppressive in GB”).

GBs synthesize G(M)-CSF

It was 25 years ago that GBs were first shown to universally stain immunohistochemically positive for GM-CSF.²⁸ GB patients have large increases in G(M)-CSF before²⁹ and after³⁰ treatment. Murine brain bearing experimental gliomas have 30 × GM-CSF of control brain.³¹

Revoltella et al.³² showed constitutively high levels of both GM-CSF and GM-CSF messenger RNA (mRNA) expression exclusively in the highest grade gliomas (World Health Organization (WHO) III and IV), and these form an element of GB cell apoptosis suppression.

G(M)-CSF is responsible for elevated neutrophil-to-lymphocyte ratio in GB

Eight independent clinical studies, two from 2015 and two from 2016, all show that peripheral blood in untreated GB shows relative lymphocytopenia, reflected by neutrophil-to-lymphocyte ratio (NLR) skewed toward neutrophils, away from lymphocytes.^33–40

Bambury et al.³⁵ in 2013 found that higher NLR in preoperative GB was associated with a faster disease course and shorter overall survival, findings independently confirmed by others in 2014³⁶ and 2016.⁴⁰ Zadora et al.³⁷ showed in 2015 that relative neutrophilia exists in de novo untreated GB compared to lower glioma grades.

Increased NLR is also seen in cancers such as non-small-cell lung cancer,⁴¹ colon,⁴² triple-negative breast,⁴³ prostate,⁴⁴ gastric,⁴⁵ hepatocellular,⁴⁶ pancreas,⁴⁷ and epithelial ovarian,⁴⁸ and other cancers. Although not a sine qua non, elevated NLR can now be recognized as a general hallmark of cancer. In 2013, Donskov reviewed the prognostic role of tumor-infiltrating neutrophils, neutrophilia, and elevated NLR and found them to be associated with poor clinical outcome in various cancers generally.⁴⁹

Neutrophilia of GB

Studies from Fossati et al.³³ and Iwatsuki et al.³⁴ indicated that glioma grade positively correlated with progressively increased glioma neutrophil infiltration and absolute peripheral blood neutrophil counts at presentation, before any drug known to generate neutrophilia (such as dexamethasone or prednisone) was given. Neutrophil activation is a poor prognostic clinical sign in GB.³⁰

Excess neutrophils have a pathophysiological trophic role of their own in driving GB growth, independent of any immunological role,^1,50–55 and as reviewed throughout this article.

Since under normal circumstances granulopoiesis is restricted to BM, GB tissue must be communicating with BM to skew NLR and generate neutrophilia. Hematopoietic BM becomes entrained with, directed by, and contributes to GB pathology.

Elevated G(M)-CSF is immunosuppressive in GB

Many factors converge to create an immunocompromised state in the course of GB. Dexamethasone and temozolomide, both commonly used during GB treatment, each contribute to a relatively lymphopenic and neutrophilic immunosuppressed state. Patients with GB exhibit systemic immune defects disproportionate to their tumor mass and disproportionate to their Karnofsky or debility score compared to other nonhematologic cancers.^56–58 Lymphopenia in GB, relative or absolute, negatively affects prognosis and success of both current standard treatment of GB with temozolomide plus radiation.^59,60

In mice subjected to controlled cortical impact, Kelso et al.¹⁵ showed that treatment with GM-CSF increased CD4⁺CD25⁺ regulatory T-cell (Treg) numbers in cervical lymph nodes coincident with decreased CNS lesion volumes and increased cortical tissue sparing. Transcriptomic analysis showed that GM-CSF induces robust immune inhibitory and neuroprotective responses 7 days following controlled cortical impact.¹⁵ Together, these results support a salutary neuroreparative role for GM-CSF in traumatic brain injury,^14,15 but a potentially deleterious effect during GB treatment. Perobelli et al. in 2016 demonstrated that G-CSF-treated neutrophils can increase Tregs with specific graft-versus-host suppressing activity, confirming earlier reports from 1995.^61,62

In 1989, Bhondeley et al.⁶³ first reported the occurrence of selective CD4⁺ lymphopenia in 10 patients with treatment-naive gliomas of different grades. The first hint into pathophysiology of this was 10 years later when Morford et al.⁶⁴ demonstrated that normal T cells exposed to glioma cell culture supernatant induced T-cell apoptosis but only with concurrent stimulation of the T-cell receptor/CD3 complex. They showed that glioma cell culture supernatant induced monocytes to release soluble factors that promoted activated T-cell apoptosis, although they could not identify the cytokine(s) responsible for this.⁶⁴ In related work, GB culture supernatants transform CD14⁺HLA-DR⁺ monocytic lineage cells into CD14⁺HLA-DR^lo/neg immune suppressors.⁶⁵

Both radiation and temozolomide act upon GB cells themselves to potentiate their innate immunosuppressive properties.⁶⁶ Supernatants from primary GB cells exposed transiently to fractionated radiation were immunosuppressive to untreated lymphocytes. These supernates of radiation-exposed glioma cells contained increased levels of many cytokines including G-CSF.⁶⁶

In the homeostatic rest state, immature myeloid cells in BM differentiate into the mature granulocytes and monocytes circulating in blood. During infection, cancer, and other homeostatic disruptions, immature myeloid cells can also differentiate into myeloid-derived suppressor cells (MDSCs).⁶⁷ MDSCs are heterogeneous, without an agreed upon marker set, but monocytic lineage MDSCs tend to be identified as CD11b+ Ly6hi LyG− and granulocytic MDSC as CD11b+ Ly6Clow Ly6G+. Elevated MDSCs are common in cancers generally^67,68 and specifically in GB.^69–75 Degree of GB infiltration with MDSC was inversely correlated with survival.⁶⁹

Increased GM-CSF content of orthotopic glioma bearing murine brain is immunosuppressive by increasing MDSC³¹ in a parallel fashion as liver metastases generate local MDSC accretions by GM-CSF oversecretion.⁷⁶

Radiation of breast cancer brain metastases lowers lymphocyte count⁷⁷ but it has never been clear how. Concomitant radiation and temozolomide after GB resection induce decreased circulating lymphocytes.⁷⁸ The most intriguing finding is the lack of an appropriate homeostatic compensatory lymphopenia-induced proliferation in these patients.⁷⁸ The remarkable conclusion is that brain tissue radiation lowers unexposed BM lymphocyte production.

Using ex vivo radiation, Kapoor et al.⁷⁹ demonstrated that radiation not only affects lymphocytes transiting the radiation field but also suppressed stem cell function in unirradiated BM. Campian et al.²¹ reported similar results in human GB/anaplastic astrocytoma where pre-radiation lymphocyte harvesting followed by reinfusing these non-irradiated lymphocytes after radiation session did not significantly ameliorate lymphopenia. G-CSF actively suppresses BM lymphocytopoiesis in mice.⁸⁰

BM responses to G(M)-CSF are biphasic, low levels reducing but higher levels increasing MDSC number and activity.³² G(M)-CSF-related immunosuppression might be particularly prominent in GB given that (1) GB occurs in a naturally G(M)-CSF-producing organ (the brain), (2) the tumor itself makes G(M)-CSF, and (3) our current mainstay treatments—resection, radiation, and temozolomide—all conspire to trigger increased G(M)-CSF.

G(M)-CSF is a direct growth factor for GB

Table 1 lists overview of G(M)-CSF growth stimulatory paths. In 2004, Li et al.⁸¹ created clones of malignant epithelial cells of C57BL/6 mice that stably expressed differing levels of GM-CSF. Clones expressing >10,000 pg/mL of GM-CSF grew faster than control, and clones expressing <100 pg/mL GM-CSF grew slower than control. Li et al.⁸¹ showed that GM-CSF was secreted by their highly producing clone induced granulocytosis and lymphopenia in vivo and was an autocrine growth factor to the clone.

Table 1.

Some of the paths by which increased G(M)-CSF can enhance GB growth.

Direct trophic effect on GB cells

Priming effect on non-transformed astrocytes for GB cells to migrate into

Increasing neutrophils that perform trophic role to growing GB and its blood supply

Decreasing BM lymphocytopoiesis

Increasing myeloid-derived suppressor cells

GB: glioblastoma; BM: bone marrow.

Orthotopic GL261 glioma–bearing mice had upregulated local production of GM-CSF.³¹ This upregulated GM-CSF turned local monocyte lineage cells (MLC) from tumor inhibiting to tumor enhancing phenotype and enhanced centrifugal migration of the malignant GB cells,³¹ findings replicated in 2016 by Kokubu et al.⁸² using a different glioma model and further demonstrating anti-GM-CSF antibody defeated this glioma-facilitating action of MLC. GM-CSF is upregulated in both human and mouse glioma microenvironments where it upregulates immunosuppressive IL-4 receptors on MDSC in that microenvironment.⁸³

Wang et al.⁸⁴ demonstrated that antibody neutralization of G-CSF inhibited growth of glioma cells in vitro. Activation of STAT3 as well as expression of several of its downstream effectors was stimulated by G-CSF contributing to the observed sustained proliferative signaling in these GB cell lines,⁸⁴ predicting benefit from dapsone, fenofibrate, and ribavirin.

Since areas around the resection cavity become ischemic after metastasis resection,⁸⁵ and residual GB cells are invariably found within 1–2 cm of an ischemic resection perimeter, these isolated pioneer cells hijack elements of the healing response, in particular G(M)-CSF increased by surgical brain tissue injury, to promote their growth in peri-resection tissue.¹⁸

Repurposed drugs reviewed in this paper, fenofibrate, ribavirin, and dapsone, have shown ability to reduce G(M)-CSF levels or consequences of G(M)-CSF (see Table 2 for an overview). If they will do so during clinical GB requires clinical research directed to answering that question.

Table 2.

Overview of the selected drugs to lower G(M)-CSF or effects of G(M)-CSF.

Drug	Marketed use	Proposed use in GB
Dapsone	Antibiotic	1. Decreased IL-8 effects 2. Reduced neutrophil contributions to GB growth
Fenofibrate	Lipid-lowering and PPAR-alpha agonist	1. Lower G(M)-CSF synthesis 2. PPAR-alpha agonism 3. Empirical anti-glioma effects
Ritonavir	Antiviral	1. Lower G(M)-CSF synthesis 2. Empirical anti-glioma activity 3. IMP inhibition

GB: glioblastoma; PPAR: peroxisome proliferator-activated receptor; CSF: colony stimulating factor; IMP: inosine monophosphate.

Dapsone

Dapsone is a 248-Da sulfone antibiotic, introduced in 1940s yet still used in treatment of leprosy and neutrophilic dermatoses like dermatitis herpetiformis or bullous pemphigoid^86–88 and as prophylaxis against malaria and Pneumocystis jirovecii (carinii). Dapsone is a small lipophilic molecule that penetrates the CNS well.⁸⁹ Antimicrobial action is by inhibition of dihydrofolic acid. Activity in cutaneous disease is due to its anti-inflammatory properties. Dapsone’s properties and mechanisms of action were recently reviewed in detail.⁸⁶ New uses continue to be discovered. In 2016, Ly et al.⁹⁰ reported steroid-sparing effect of dapsone in treating giant cell arteritis. We go into detail on dapsone’s mechanism of action in the neutrophilic dermatoses due to its applicability to dapsone’s intended use in GB.

In bullous pemphigoid, immunoglobulin is deposited in skin, producing leukocyte chemo-attractants at the site of antibody fixation, resulting in neutrophil accumulation at the site of damage.^87,91 Dapsone does not influence the primary pathology deposition of antibodies in the skin,^87,88 but instead reduces the destructive consequences of neutrophil infiltration by reducing neutrophil chemotaxis and adhesiveness.^88,91–93

IL-8 is sequestered on endothelia. Neutrophils move along an IL-8 gradient.^94,95 IL-8 also activates neutrophils, increasing surface expression and binding activity of MAC1 (a complement receptor).^96,97 In patients with dermatitis herpetiformis, serum IL-8 is elevated following the immune response to dietary gluten in the small bowel. In active skin lesions, IL-8 mRNA and neutrophil CD11b expressions are increased.⁹⁸ Therapeutic levels of dapsone reduce IL-8 and stop immunoglobulin G-induced IL-8 release from cultured keratocytes.⁹² Dapsone also stops IL-8 secretion from lipopolysaccharide-stimulated human bronchial epithelial cells.⁹⁹ In skin section assays, therapeutic dapsone levels inhibited neutrophils’ adhesion to epidermis⁹³ or basement membrane bound antibody.⁹¹ Dapsone thus inhibits neutrophil accretions, migration, and response to IL-8, and production of IL-8—all potentially useful attributes in treating GB.

Repression of endogenous IL-8 production in GB cells or addition of IL-8 neutralizing antibodies reduce GB proliferation and invasion.^100,101 IL-8 also has an autocrine aspect, since GB cells also express the IL-8 receptor CXCR-1. Antibody-mediated inhibition of CXCR-1 reduces GB cell invasiveness.¹⁰⁰

Standard radiation of the post-resection GB tumor bed induced an increase in IL-8 expression in radiation-exposed brain tissue, setting the stage for enhanced growth of surviving GB cells¹⁰² given that increasing IL-8 enhances GB growth and motility.^103,104 Heavier IL-8 staining in biopsied human GB tissue is associated with greater microvascular proliferation and shorter overall survival.^105,106

In GB, a subpopulation of cancer stem-like cells (CSCs) exists adjacent to endothelial cells in a perivascular niche.¹⁰⁴ These endothelial cells increase IL-8 secretion on encountering neutrophils’ IL-8, enhancing endothelium’s growth, forming a feed-forward amplification cycle with neutrophils.^51,105 IL-8 is increased around areas of GB necrosis, driving centrifugal GB cell migration and neutrophil accretions here.^100,103,106 Indeed, higher neutrophil count during standard GB treatment is a poor prognostic sign.¹⁰⁷

Dapsone’s ability to cross the blood–brain barrier combined with its inhibitory effect on IL-8 production, neutrophil accretion, and neutrophil homing make it a strong candidate for potential adjunctive GB treatment.

Fenofibrate

Fenofibrate is a 361-Da drug, marketed since the early 1980s as a lipid-lowering agent. It is also a peroxisome proliferator-activated receptor alpha (PPAR-alpha) agonist.¹⁰⁸ Fenofibrate diminished IL-1 beta stimulated increases in G-CSF and GM-CSF in airway epithelia.¹⁰⁹ In studying the antiatherogenic effects of fenofibrate in mice fed with a high cholesterol diet, Kooistra et al.¹¹⁰ found decreased aortic wall GM-CSF, along with decreased levels of other inflammatory mediators.

Fenofibrate has anti-glioma effects also via PPAR-alpha agonism^111–114 and by related adenosine triphosphate (ATP) depletion in glioma cells.¹¹³ Empirical evidence indicated a fenofibrate-mediated decrease in stemness mediators CD133 and transcription factor Oct4 in glioma cell populations.¹¹⁴

A seven-drug cocktail to treat a G-CSF producing tumor-progressive medulloblastoma using fibrate is in current clinical trial in Austria (ClinicalTrials.gov Identifier: NCT01356290, the MEMMAT regimen). MEMMAT uses four adjuvants—bevacizumab, thalidomide, celecoxib, and fenofibric acid—to augment three traditional cytotoxic drugs—etoposide, cyclophosphamide, and cytarabine.

Ribavirin

Ribavirin is a 244-Da triazole guanosine analog with a half-life of several days. Its antiviral activity made it a cornerstone of the now outmoded treatment of hepatitis C virus (HCV). Ribavirin remains useful in treating human respiratory syncytial virus infections. The mechanism of action of ribavirin’s antiviral activity is not fully understood, although five major mechanisms of action are proposed:^115–118

Immunostimulation by upregulating cytokines to shift T helper (Th) ½ cell balance to Th1 dominance.

Inhibition of 24-kDa eukaryotic translation initiation factor 4E (eIF4E) function, thereby inhibiting mRNA capping.

Modulation of interferon-alpha-related gene expression.

Direct inhibition of inosine monophosphate dehydrogenase with consequent depletion of intracellular guanosine triphosphate (GTP).

After triphosphorylation, ribavirin triphosphate is incorporated into replicating RNA viral RNA polymerases with consequent induction of viral mutagenesis.

Two studies from 2016 show significant glioma cell killing by ribavirin^119,120 confirming a 2014 study showing ribavirin-induced G0/G1 arrest in seven glioma cell lines at median 55 microM IC50 (range = 28–664).¹²¹ Higher platelet-derived growth factor receptor expression, a major driver of GB growth,¹²² predicted lower IC50 of ribavirin.¹²¹ Usual plasma levels during HCV treatment are 2500 ng/mL (~10 µM).¹²³

Neutropenia is the most frequent side effect of treatment with ribavirin. Liu et al.¹²⁴ found that HCV patients treated with ribavirin have lower serum levels of G-CSF and GM-CSF. Ribavirin drives neutropenia by several mechanisms, among which G(M)-CSF suppression is primary.

Ribavirin shows dose-dependent growth inhibition with G_0/1 arrest.¹²¹ The related inosine monophosphate dehydrogenase inhibitor tiazofurin shows decreased extracellular vascular endothelial growth factor (VEGF) levels¹²⁵ in GB cell lines, but also shows increased glial fibrillary acidic protein and increased morphologic cell differentiation.¹²⁶

Conclusion

Data presented in this article lead to eight salient points:

Normal CNS neurons, normal CNS glia, and GB cells synthesize G(M)-CSF.

G(M)-CSF plays important roles in normal CNS homeostasis, neuronal function, pruning, repair, neurite outgrowth, and neurodevelopment.

Excessive brain-synthesized G(M)-CSF diverts BM hematopoiesis away from lymphocyte lineage toward granulocyte/MLC, creating a lymphopenic–neutrophilic state.

Brain tissue trauma of any origin—blunt, sharp, ischemic, infectious, cytotoxic, tumor encroachment, or radiation—increases synthesis of G(M)-CSF in brain.

This contributes to the neutrophilia, lymphopenia, and reduced immunocompetence in GB, stroke, whole brain radiation treatment, and traumatic brain injury.

Three older drugs, dapsone, fenofibrate, and ribavirin, have ancillary attributes with potential to lower G(M)-CSF levels or effects. However, significant work must be done to validate these drugs specifically in GB to confirm that they are indeed viable GB treatment adjuncts.

GB is a vascular tumor where IL-8 drives hypervascularization, proliferation, and migration. Dapsone inhibits IL-8 production and the GB growth enhancement by neutrophils.

GB is a systemic, whole body disease, as Stromnes et al.¹ and others have pointed out as a feature of cancers generally. This article gives evidence of how GB communicates with BM to promote growth via G(M)-CSF.

This article provides substance and mechanistic elements on how neutrophils and tumor-secreted G(M)-CSF contribute to GB proliferation, angiogenesis, and immunosuppression. Three older drugs—dapsone, fenofibrate, and ribavirin—might reverse some of these contributions.

Footnotes

Acknowledgements

This article came about during the authors’ discussions over the last 2 years under the aegis of the International Initiative for Accelerated Improvement of Glioblastoma Care, The IIAIGC Study Center. All authors contributed equally and have read and approved the final manuscript. This was an unfunded research.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The author(s) received no financial support for the research, authorship, and/or publication of this article.

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